Key elements of tumorigenesis are driven by paracrine signaling between stromal and epithelial cells within the context of the extracellular matrix. Unfortunately, the cellular assays used for drug discovery typically rely on a single cell type grown as a monolayer, and this aspect of tumor biology very poorly. This may be one factor contributing to the low success rate of anti-cancer drugs in clinical trials. Though cellular models incorporating multiple cell types in a tissue-like environment are available, they are difficult to integrate with current automated microscopic imaging platforms used by pharma for high content analysis of cell function. An especially challenging problem is detection of soluble paracrine signaling factors in extracellular matrix. Though antibodies are available for many of these factors, existing immunoassay methods are not suited for use in a dense matrix. A fundamental limitation is the requirement for multiple components: the primary antibody and secondary reporter reagents. To overcome this limitation we are proposing to develop an intramolecular immunoassay;i.e., an antibody with a built-in fluorescent reporter. We will achieve this by tethering a fluorescent tracer on a flexible polypeptide linker to IgG and Fab fragments engineered with seleno cysteine residues for site specific modification. A well characterized monoclonal antibody for vascular endothelial growth factor (VEGF) will be used as an initial model. A series of prototype intramolecular immunoassay reagents will be assembled and tested for fluorescence based detection of VEGF to arrive at the optimal structure. In Phase II, the intramolecular immunoassay will be combined with BellBrook's novel microconduit array platform, iuvo, to enable dynamic imaging of paracrine signaling at the level of soluble factors secreted from individual cells. PUBLIC HEALTH RELEVANCE: Important aspects of tumorigenesis are controlled by signaling between different cell types that reside in a dense matrix that provides structural support to tissues and also participates in signaling. Unfortunately, it is very difficult to incorporate these signaling events into in vitro assays that can be used to test potential anti-cancer drugs. To overcome this limitation, we propose to develop a novel immunoassay method that would allow probing of cell-cell signaling in a tissue-like matrix using existing automated detection instruments.